Queuosine biosynthesis is required for sinorhizobium meliloti-induced cytoskeletal modifications on HeLa Cells and symbiosis with Medicago truncatula.

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton

Cytoskeleton modifications of HeLa cells infected with <i>S. meliloti</i> queuosine biosynthesis mutants.

<p>HeLa cells were incubated with <i>S. meliloti queC</i> (A, E), <i>queF</i> (B, F), <i>tgt</i> (C, G), <i>queA</i> (D, H), the wild type 1021 strain (I) and the <i>queF</i> complemented strain (GMI11186) (J) in 0.5% FCS culture medium alone (A–D, I, J) or supplemented with preQ1 (E–H). HeLa cells were stained with phalloidin-Texas red and observed by fluorescence microscopy 48 hpi. Scale bar: 10 µm.</p

Bacteria-induced cytoskeleton modifications of HeLa cells.

<p>HeLa cells untreated (A), inoculated with <i>S. meliloti</i> (B), <i>R. leguminosarum</i> (C), <i>A. caulinodans</i> (D), <i>C. taiwanensis</i> (E), <i>B. tuberum</i> (F), <i>C. crescentus</i> (G) and <i>E. coli</i> (H). HeLa cells were stained with phalloidin-Texas red and observed by fluorescence microscopy 48 hours after bacterial inoculation. Arrow: stress fiber.</p

Bacterial strains and plasmids.

a<p>The location of plasmid insertions (number of nucleotides after the start codon) is indicated between brackets.</p

The <i>S. meliloti</i> queuosine biosynthetic pathway.

<p>preQ₀: 7-cyano-7-deazaguanine, preQ1: 7-(aminomethyl)-7-deazaguanine, AdoMet: S-adenosyl-L-methionine, EpoxyQ: epoxyqueuosine, Q: queuosine. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056043#pone.0056043-IwataReuyl1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056043#pone.0056043-Reader1" target="_blank">[50]</a>.</p

Symbiotic phenotype of <i>S. meliloti</i> queuosine mutants.

<p>Dry weight of <i>M. truncatula</i> seedlings inoculated with <i>S. meliloti</i> 1021, different queuosine-deficient mutants and the <i>queF</i> complemented (GMI11186) strain at 40 dpi. Statistical significance (P<0.01) is shown with respect to strain 1021(*) and the <i>queF</i> mutant (^#), respectively. (B, C) Sections of <i>M. truncatula</i> 21–day old nodules induced by 1021 (B) and the <i>queF</i> isogenic mutant (C). (D, E) Electron micrographs of nodule cells infected with 1021 (D) or the <i>queF</i> mutant (E). <i>queF</i> mutant bacteroids are randomly organized within the infected cell whereas 1021 bacteroids show a radial organization. (Insert panel in E): arrows point to symbiosome membranes detached from <i>queF</i> bacteria (). Arrowhead, type 4/5 bacteroid. *, starch granules.</p

Determination of GTPases activation state in bacteria-treated HeLa cells.

<p>(A) Representative pull down assays of active Cdc42, Rac1 and RhoA GTPases at 48 hpi in non-inoculated- (control), <i>S. meliloti</i> 1021- and <i>queF</i>-inoculated HeLa cells. (B) Quantification of pull down assays using ImageJ software. Means ± S.D. were calculated from three independent experiments for Cdc42-GTP and mean from two independent experiments for Rac1-GTP and RhoA-GTP. Results were normalized to the corresponding total protein. Statistical significance (P<0.001) is shown (*) with respect to the control. (C) Immunoprecipitation of active and total CdC42 from non-inoculated (control) HeLa cells or cells inoculated with live and heat-killed wild-type bacteria 48 hpi. (D). Kinetics of Cdc42 activation. Actin, total Cdc42, Rac1 and RhoA or active GTP-bound forms of Cdc42, Rac1 or RhoA were detected by immuno-blotting of SDS-PAGE gels.</p